Silicon and silicon-based nanoparticles (SiNP) attract scientific attention due to the biocompatibility and assimilation of silicon by body tissues. Iron-doped SiNP (SiFeNP) allow the use of ferromagnetic properties of iron for NP detection and the possibility of therapeutic application of SiFeNP. The purpose of this work was to analyze the interaction of SiFeNP with epithelial cells (EC) COLO357 and SW620 and human peripheral blood lymphocytes (PBL). SiFeNP were obtained by laser ablation and divided into the NP1 and NP2 fractions of 100 and 150 nm size, respectively. Cytotoxicity, apoptosis induction, reactive oxygen species (ROS) production, and lysosome metabolism were analyzed using in vitro methods. EC were found to efficiently incytosed both types of NPs, which resulted in the increase in the granularity of cells. NP did not cause apoptosis or EC necrosis, but accumulated in lysosomes, which led to a decrease in the membrane potential of lysosomes. In turn, a decrease in the level of EC metabolism led to a gradual (24 h) increase in ROS production by 10–15%. NP1 caused more ROS than NP2, and accumulated more in the EC, which may be the result of a difference in the particle size. SiFeNP did not interact with PBL. Thus, the total cytotoxicity of SiFeNP did not exceed 20%, which is associated with a decrease in lysosome metabolism and insignificant ROS production.